Chapter 2 Centrifugation:

Separation of Organelles and Biomolecules

Biochemistry and Molecular Biology (BMB)

3.1 Introduction 3.2 3 2 Basic Principle of sedimentation 3.3 Types, care and safety of centrifuges 3.4 3 4 Preparative centrifugation (example on influenza virus and protein complex) 3.5 3 5 Analytical centrifugation
Analytical Biochemistry A l ti l Bi h i t (AB)

3.4.3 Ultracentrifugation
Koolman, Koolman Color Atlas of Biochemistry (CAB), 2nd edition (CAB)
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General Steps in Biochemical Separation

Separation of Macromolecules (CAB)

Chromatography, precipitation Ch t h i it ti Electrophoresis, ultracentrifugation

Introduction (MBM 3.1) Principles of centrifugation ()

A centrifuge is a device for separating particles from a solution according to their size shape density viscosity size, shape, density, of the medium and rotor speed
In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top. The greater the difference in density, the faster they move If there is no difference in move. density (isopyknic conditions), the particles hover. To take advantage of even tiny differences in density t separate various diff i d it to t i particles in a solution, gravity can be replaced with the much more powerful centrifugal force provided by a centrifuge.
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Type 1: Analytical Centrifugation Applications: Measure the shape or mass of dystroglycan supermolecular molecules In skeletal muscle,dystrophin- is a component of the

glycoprotein complex (DGC)(Fig 1) (DGC)(Fig. 1). Dystroglycan is an extracellular peripheral membrane glycoprotein anchored to the cell membrane by binding to a transmembrane glycoprotein, -dystroglycan. The 1. dystroglycan dystroglycan dystroglycan- -dystroglycan complex is widely expressed in a broad array of tissues and is thought to stabilize the p plasma membrane by acting as an y g axis through which the extracellular matrix is tightly linked to cytoskeleton. y gy gy This is because -dystroglycan strongly binds to laminin in the extracellular matrix, and the cytoplasmic domain of -dystroglycan interacts with dystrophin, which in turn binds to the actin http://www.cgmh.org.tw/chldhos/intr/c4a90/new_page_50.htm cytoskeleton2. 5

Type 2: Preparative Centrifugation

Application: Separation of cell, subcellular structure cell structure, membrane vesicles

Lipid rafts ()

Lipid rafts () are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms. Rafts are receiving increasing attention as devices that regulate 6 membrane function in eukaryotic cells.

Raft Proteins Are Targets fro Disease

Simons, K. et al. J. Clin. Invest. 2002;110:597-603

Alzheimer disease Parkinson disease Hypertension

3.2 Basic Principle of Sedimentation (AB 3.4.3)

Centrifugal force
F = M 2 r

M: mass of particle r: radius of rotation (cm) (ie distance of particle f di f i l from axis i of rotation) :Average angular velocity A l l i (radians/sec) 2 rev min -1 = 60 Rev: revolution per minute (r.p.m.) 1 revolution = 2 radians l ti 2 di 8 =360

Centrifugal Field
G=r2
depends on the radical distance of the particle from the rotation axis and the square of the angular velocity

G=

(rev min ) r
-1 2

3600

Angular Velocity
2 rev min -1 = 60
rev: revolution per minute (r.p.m.) (r p m )

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Relative centrifugal force (different radius of rotor)

RCF value f M r RCF = c = = 2 r g -1 /, "No. x g" fg Mg ( (multiples of earth's g p gravitational force) ) 2 (). 2 rmp -1
2

RCF =

60

rg

RCF = 1.12 x 10-5 x (rpm)2 x r

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Relative centrifugal force

RCF = C 1.12 x 10-5 x (rpm)2 x r

rmin rmax

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Interacting Forces in Centrifugation

Sedimenting force, M2r, is opposed by...
1. Frictional Resistance against particle moving through fluid. fluid = f.v f = frictional coefficient of particle in the solvent v = particle velocity

Fcentrifuge

Ffriction + Fbuoyancy

2. Flotation Force F = Ms

2r

Ms = the mass of equal volume of solvent BALANCE between the sedmenting force and counteracting force Net force =

(M

-M s ) r - fv M
2

M, f: relate to the mass and shape of analyte

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Sedimentation Coefficient (s),

()

w 2r(mp-ms) - fv = 0 Rate f S di R t of Sedimentation t ti

Svedberg unit
Theodor Svedberg(1884-1971) 1926 Nobel prise 1908,
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Fcent = M r
2

M : mass

: angular vel it l locity

r : distance form center of rotation : solvent density vb : particle specific volume of particle p p p f : frictional coefficient v : velocity of particle

Fbuoy = M r vb
2

F frict = fv Fcent = Fbuoy + F frict v = M r (1 v ) / f

2

S = v / r
2

S:

S = M (1 v b ) / f
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M= particle mass f = frictional coefficient of the particle in the solvent = d density of solution i f l i v = particle velocity 2 particle specific volume (cm3/g ) /g, S is increased for particle of larger mass (because sedimenting f (b di ti force a M(1 ) M(1-vr) S is increased for particle of larger density (equal volume) S is increased for more compact structures (Shape) of equal particle mass (frictional coefficient is less) S is increased with rotational speed Mild, non-denaturing procedure is useful for protein purification, and f intact cells and organelles ifi ti d for i t t ll d ll
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M= particle mass p f = frictional coefficient of the particle in the solvent = density of solution v = particle velocity 2 particle specific volume (cm3/g, ) 2 <1 (>) , 2 =1 (=) , ,

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Separation by Sedimentation
Weight Material
100 kg
30 kg 10 kg 10 kg 8
1

Iron

Stone

Iron

Stone

Cotton

Iron

Higher den nsity

Sedim mentation

Mass M Density
10 kg

30 kg 10 kg
1

Shape

100 kg

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Densities and sedimentation coefficients for biomolecules, cell organelles, and viruses.

Require high density media High concentrated g CsCl

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Sedim mentat tion

Soluble S l bl protein DNA RNA

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NOMOGRAMS
Conversion between relative centrifugal force
Equation used to calculate NOMOGRAMS (BMB Fig. 3.1) for quickly finding RCF at given speed and rotor type (radius). (radius)

Radical distance (mm)

Relative centrifugal field (xg)

22 Rotor speed (r.p.m)

Types of Centrifuge BMB 3.3.1

Maximum speed of sedimentation Presence /absence of vacuum Temperature control refrigeration) Volume of sample and capacity of centrifugation tubes

Microfuge
0.5-1.5 cm3, 10,000 g Concentration of protein samples

Large-capacity preparative centrifuge

5-250 5 250 cm3, 3,000-7,000 3 000 7 000 g

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High-speed refrigerated centrifuge g p g g

5-250 cm3, 100,000 g Differentiation separation of nucleus, Diff ti ti ti f l mitochondrial, protein precipitate, large intact organelle, cellular debris organelle

Ultracentrifugation
5-250 cm3, 600,000 g , Microsomal vesicles, ribosome Has to reduce excessive rotor temperature generated by frictional resistance sealed chamber, evacuated, cooling
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Centrifuge Rotors
Fixed Angle Rotor

(MBM3.3.2)

Swinging Bucket Rotor

Sedimenting particles have only short distance to travel before pelleting. Shorter run time. The Th most widely used rotor t t id l d t type.

Longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet.

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Centrifuge Rotors
Fixed Angle Rotor

(MBM3.3.2)

Vertical Tube Rotor

Swinging g g Bucket Rotor

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Centrifuge Its Use and Safety (BMB 3 3 4) 3.3.4)

On December 16, 1998, milk samples were running in a Beckman L2-65B ultracentrifuge L2 65B using a large aluminum rotor . The rotor failed due to excessive mechanical stress

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Mechanical stress
Always ensure that loads are evenly balanced before a run. Always observe the manufacturers maximum speed and sample density ratings. Always observe speed reductions when running high density solutions, plastic adapters, or stainless steel t b l ti d t t i l t l tubes.

Corrosion
Many rotors are made from either titanium or aluminum alloy, chosen for their advantageous mechanical properties. While titanium alloys are quite corrosion-resistant, aluminum alloys are not. When corrosion occurs, occurs the metal is weakened and less able to bear the stress from the centrifugal force exerted during operation. The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress levels than an uncorroded rotor
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Differential Centrifugation

BMB 3.4.1

Based on the differences in the sedimentation rate of the biological particles of different size, shape and density

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Moving Zone (differential) Centrifugation -Rate Rate

Incomplete sedimentation (lower operation speed). Control time and solution densit sol tion density Mostly used for separation with similar density and different size/shape
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Moving Boundary (differential) Centrifugation

1) 2) 3)

1) The entire tube is filled with sample and centrifuged 2) Through centrifugation, one obtains a separation of two particles but any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation diti f t if ti 3) Repeat sedimentation at different speed

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Moving Boundary (differential) Centrifugation

Advantages: Large-scale preparation Disadvantages: Disad antages Poor resolution. Poor purity Difficult to separate analytes with similar sedimentation coefficient

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Density Gradient Centrifugation (BMB 3.4.2)

Important technique for purifying proteins and particularly nucleic acids. d ti l l l i id
Two different types of density gradient centrifugation, for two different purposes are: p p Zonal (or Rate Zonal) Centrifugation (Sucrose density gradient centrifugation) Iso-density (Isopycnic) Centrifugation (Caesium chloride density gradient centrifugation)
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Moving Zone (differential) Centrifugation -Rate Rate

1) 2)

1) Sample is applied in a thin zone at the top of the centrifuge tube on a density gradient solution 2).Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, 35 shape and density

Iso-density (Isopycnic) Centrifugation

-equilibrium

(AB3.4.3)

Isopycnic = same density = density equilibrium between analyte and solution Both analytes and solution have different densities

1) Preparation of solution with different densities E.g. sucrose : Good G d water solubility f making hi h t l bilit for ki high concentration of solution Cheap
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Iso-density (Isopyncic) Centrifugation

-equilibrium

(AB3.4.3)

2). molecule floats or sinks to position where density equals density of CsCl solution Then no net sedimenting force on molecules solution. molecules. and separation is on basis of different densities of the particles.

Molecules separated on equilibrium position, NOT by O 37 rates of sedimentation.

Comparison of Two Methods

Moving Zone Centrifugation Isopyncic centrifugation

Centrifugation:

Lower speed, not complete sedimented, stop at proper time Sedimentation Rate

Completely sediment to where the density is equilibrated, high speed, speed long running time Sedimentation equilibrium Similar MW, different density Nucleic id N l i acid / cell organelle
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Sample:

Similar density, different MW/shape Protein ( i il density, P t i (similar d it but different in MW)

Density Gradient Centrifugation

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The Influenza A Virus

Studded in the lipid bilayer are two integral membrane proteins some 500 molecules of hemagglutinin ("H") and d some 100 molecules of neuraminidase ("N")

Within the lipid bilayer are some 3000 molecules of matrix protein 8 pieces of RNA Each of the 8 RNA molecules is associated with a globular particle (about 100 nm in diameter) sheathed in a lipid bilayer (derived from the plasma membrane of it h t) l b f its host) (1) many copies of a nucleoprotein i f l t i (2) several molecules of the three subunits of its RNA polymerase (3) some "non-structural" protein molecules of uncertain function

Virus Purification Four commonly used methods

Differential centrifugation and density gradient centrifugation di t t if ti Precipitation of viruses Denaturation of contaminants Enzymatic digestion of cell constituents

How to purify virus?

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Purification of influenza A virus (H1N1) by Density Gradient Centrifugation

1. After 48 hours of incubation the allantoic fluid containing virus was purified by low speed f centrifugation (4000 rpm 40 min). 2. Virus was pelleted by high speed centrifugation at 18000rpm for 1.5 h in the SW27 rotor. The virus pellet was collected, diluted in STE buffer and layered over linear 30 to 60 % sucrose gradient prepared in STE buffer. -----Isopyconic gradient centrifugation:(): 3. Sample recentrifuged at 25000 rpm i th SW27 3 S l t if d t in the rotor for 2.5 h at 4 C, then the virus band was collected, diluted with STE buffer and pelleted by centrifugation at 30000 rpm for 1.5 h at 4 The C. pellet of 4. virus was collected and diluted with STE buffer. BioMarket Ltd. www.biomarket.fi

Virus Visualization by Electron Microscopy

100nm

Sarcolemma ( : It is the surface membrane of the entire fiber

Skeletal Muscle

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Sarcolemma :It is the surface membrane of the entire fiber (, ( ) T-tubular membranes They contain extracellular fluid (high in Ca and Na ions) They are continuous tubes of sarcolemmal membrane that run through (transversely) the muscle fiber. Sarcoplasmic reticulum ()-\ The Th sarcoplasmic reticlum (SR) is the C store. l i ti l i th Ca t It is a diffuse membrane structure that surrounds the sarcomere 46

How can we do separation?

Step 1

Step 2

Step 3

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Step 1- Cell Homogenization ()

To obtain pure organelles, the cells must be ruptured, so that the cell membrane is broken, but the organelle to be studied is not. The process of rupturing a cell i k f t i ll is known as homogenization of the cell and the subsequent isolation of organelles is called fractionation.48

Four Common Methods

Using gentle mechanical procedures, called homogenization, the g , plasma membranes of cells can be ruptured so that the cell contents are released. 1. Sonication 2. Detergent lysis 3. 3 French press 4. Mechanical homogenization h i i
49

Ruptured cells producing a liquified cellular homogenate

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Step 2-Cell Fractionation by Centrifugation.

(1)

(2)

(3) (4)

Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component the general component, 51 greater is the centrifugal force required to sediment it.

Step 3- Density Gradient Centrifugation

Sarcolemma

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Step 4 Collection f F St 4- C ll ti of Fractions ti

Manual collection by pipette Automatic fraction collector for unstable gradient Freezing and slicing

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Contractile Apparatus of Muscle

Electron g p micrographs of individual myosin protein molecules

Myosin is a major component of the contractile apparatus of muscle. As shown here, it is composed of two globular h d regions li k d t a common ft l b l head i linked to rodlike tail.

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Hydrophobic and Low Abundant Membrane Proteome

Immuno-modulation, Molecular recognition , Cell surface adhesion Heterogeneous Glycosylation
P P P P P P P P P P P P P P

Dynamic, Spacious Complexity of Phosphorylation

Cell Cycle

Signal transduction

Differentiation, proliferation

Protein degradation Apoptosis

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P ifi

ti

di

d lt

t if

ti

Affinity Purification of Membrane y Vesicles (BMB 3.4.5)

In-side-out Right-side-out Cross-contamination of vesicular membrane protein Inside-out vesicles, right-side-out vesicle, membrane sheet, leaky vesicles Smaller vesicles are trapped in large vesicles In-side-out (cytoplasmic side out) ( y p ) Right-side-out (apoplastic side out) 56 vesicles

Lectin Agglutination Method (by Lectin carbohydrate Interaction) Lectin-carbohydrate

Lectin: protein that interact with carbohydrate There are many carbohydrates on the surface of cell
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No carbohydrate Inside-out: No carbohydrate I id t N b h d t

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Lectin Agglutination Method

WGA: Wheat germ agglutinin SL: Sarcolemma SN: supernatant No carbohydrate

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Immunoblot Analysis for Verification of Different Subcellular Fractions

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Analytical Ultracentrifugation (AUC)

MBM 3.5.1 351

An analytical ultracentrifuge spins a rotor at an accurately controlled speed and temperature. The concentration distribution of the sample is determined at known times using absorbance measurements. It can determine: Relative molecular mass of solute Purity of macromolecule Change in relative molecular mass of supermolecular g p complexes Shape, Conformational change of protein structure Ligand-binding study

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Optical System of an Analytical Ultracentrifugation

(Beckman Optima XL-A):

back to top

This figure displays a schematic diagram of the Beckman Optima XL-A absorbance system. A high intensity xenon flask lamp allows th use of fl k l ll the f wavelengths between 190 and 800nm. The lamp is fired briefly as a selected sector passes the detector.

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Sedimentation Velocity Method

Mass, Shape and Conformation; Higher velocity

Sedimentation velocity experiments Sedimentation are performed at high speed to overcome the effect of diffusion. For a sedimentation velocity experiment, an di i l i i initially uniform solution is placed in a cell and a sufficiently high angular velocity is applied to cause rapid sedimentation of solute towards the cell bottom. As a result, there is a , depletion of solute near the meniscus, causing a characteristic spectrum as shown in the following figure A sharp figure. boundary occurs between the depleted region and the sedimenting solute (the plateau)
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Diffusion

Determination of Sedimentation Coefficient (s)

4000 s 6000 s 8000 s 10000 s

The velocity of the individual particles in SV experiments cannot be resolved, but the rate of movement of the boundary region can be measured. measured From this, the sedimentation coefficient (s) can be this determined. Remember, s depends directly on the mass of the solute particles and inversely on the frictional coefficient, which is a measure of size of the solute particles particles.
http://www-bioc.rice.edu/bios576/AU/AU_Page.html#au
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Sedimentation Equilibrium Methods

Mass, Complex formation; Lower velocity
Sedimentation equilibrium S di t ti ilib i experiments have a lower rotor speed than sedimentation velocity experiments. Solute particles do not pellet at the bottom of the cell, but instead the process of diffusion opposes the process of sedimentation until after a period of time, the two opposing forces reach equilibrium and the apparent concentration profile does not change. At equilibrium, the concentration of the solute increases exponentially towards the cell bottom. Each column displays a different absorbance p profile, because the , concentrations of sample are 65 varied in each.

Sedimentation Analysis of Supramolecular Protein Complex

The binding of ligands may induce conformational changes i subunits of f ti l h in b it f biomolecules, which changes the supramolecular structure of complex.

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